U.S. patent number 5,484,782 [Application Number 08/442,473] was granted by the patent office on 1996-01-16 for (e)-20(22)-dehydrovitamin d compounds.
This patent grant is currently assigned to Wisconsin Alumni Research Foundation. Invention is credited to Hector F. DeLuca, Rafal R. Sicinski.
United States Patent |
5,484,782 |
DeLuca , et al. |
January 16, 1996 |
**Please see images for:
( Certificate of Correction ) ** |
(E)-20(22)-dehydrovitamin D compounds
Abstract
Vitamin D.sub.3 analogs in which a double bond has been
introduced into the side chain between carbons 20 and 22. The
compounds are characterized by a marked intestinal calcium
transport activity while exhibiting much lower activity than
1.alpha.,25-dihydroxyvitamin D.sub.3 in their ability to mobilize
calcium from bone. Because of their preferential calcemic activity,
these compounds would be useful for the treatment of diseases where
bone formation is desired, such as osteoporosis. The compounds also
have relatively high HL-60 cell differentiation activity making
them useful for the treatment of diseases characterized by abnormal
cell differentiation or cell proliferation, such as psoriasis.
Inventors: |
DeLuca; Hector F. (Deerfield,
WI), Sicinski; Rafal R. (Warsaw, PL) |
Assignee: |
Wisconsin Alumni Research
Foundation (Madison, WI)
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Family
ID: |
22522410 |
Appl.
No.: |
08/442,473 |
Filed: |
May 16, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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147663 |
Nov 3, 1993 |
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Current U.S.
Class: |
514/167;
552/653 |
Current CPC
Class: |
A61P
43/00 (20180101); C07J 71/0042 (20130101); C07C
401/00 (20130101) |
Current International
Class: |
C07C
401/00 (20060101); C07J 71/00 (20060101); C07C
401/00 (); A61K 031/59 () |
Field of
Search: |
;552/653 ;514/167 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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184112 |
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Jun 1986 |
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EP |
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221776 |
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Nov 1989 |
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GB |
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WO89/10351 |
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Nov 1989 |
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WO |
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WO89/10352 |
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Nov 1989 |
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WO |
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Other References
Perlman et al., "24-Homologated 1,25-Dihydroxyvitamin D.sub.3
Compounds: Separation of Calcium and Cell Differentiation
Activities", Biochemistry, vol. 29, No. 1, pp. 190-196, 1990. .
Murayama et al, "Synthesis and Immunoregulating Activity of Vitamin
D Analogues Bearing Pregnane Side Chains", Bioorganic and Medical
Chemistry Letters, vol. 2(10), pp. 1287-1292, 1992. .
Murari et al, "Synthesis and Biological Activity of
3.beta.-Hydroxy-9,10-Secopregna-5,7,10[19]-Triene-20-One: A Side
Chain Analogue of Vitamin D.sub.3 ", Journal of Steroid
Biochemistry, vol. 17, pp. 613-619, 1982. .
Matoba et al, "Structural Modification of Bioactive Compounds. I.
Syntheses of Vitamin D Analogues I.", Chemical and Pharmaceutical
Bulletin, vol. 32, No. 4, Tokyo, Japan, pp. 1416-1422, Apr. 1984.
.
Perlman et al, "20-Oxopregnacalciferols: Vitamin D. Compounds that
Bind the Progesterone Receptor", Tetrahedron Letters, vol. 35, No.
15, Oxford, Great Britain, pp. 2295-2298, Apr. 1994..
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Primary Examiner: Kestler; Kimberly J.
Attorney, Agent or Firm: Andrus, Sceales, Starke &
Sawall
Government Interests
This invention was made with United States Government support
awarded by the National Institutes of Health (NIH), Grant No.
DK-14881. The United States Government has certain rights in this
invention.
Parent Case Text
This application is a divisional of copending application Ser. No.
08/147,663 filed Nov. 3, 1993.
Claims
We claim:
1. A compound having the formula ##STR11## where Y.sup.1 is
hydrogen or a hydroxy-protecting group, R.sup.1 represents
hydrogen, hydroxy or protected hydroxy, and where R is hydrogen,
aryl, alkyl, hydroxyalkyl or fluoroalkyl group, or R may represent
the following side chain fragment: ##STR12## wherein R.sup.1 is as
defined above, R.sup.2 and R.sup.3 are each selected from the group
consisting of alkyl, hydroxyalkyl and fluoroalkyl, or, when taken
together represent the group --(CH.sub.2).sub.m -- where m is an
integer having a value of from 2 to 5, R.sup.4 is selected from the
group consisting of hydrogen, hydroxy, fluorine, O-acyl, alkyl,
hydroxyalkyl and fluoroalkyl, R.sup.5 is selected from the group
consisting of hydrogen, fluorine, alkyl, hydroxyalkyl and
fluoroalkyl, and wherein n is an integer having a value of from 1
to 5.
2. (E)-20(22)-dehydro-1.alpha.,25-dihydroxyvitamin D.sub.3.
3. A pharmaceutical composition containing at least one compound as
claimed in claim 1 together with a pharmaceutically acceptable
excipient.
4. The pharmaceutical composition of claim 3 containing
(E)-20(22)-dehydro-1.alpha.,25-dihydroxyvitamin D.sub.3 in an
amount from about 0.5 .mu.g to about 50 .mu.g.
Description
BACKGROUND OF THE INVENTION
This invention relates to biologically active vitamin D compounds.
More specifically, the invention relates to
(E)-20(22)-dehydrovitamin D compounds, to a general process for
their preparation, and to their use in treating osteoporosis and
psoriasis.
With the discovery of 1.alpha.,25-dihydroxyvitamin D.sub.3 as the
active form of the vitamin has come an intense investigation of
analogs of this hormonal form of vitamin D with the intent of
finding analogs that have selective activity. By now, several
compounds have been discovered which carry out the differentiative
role of 1,25-dihydroxyvitamin D.sub.3 while having little or no
calcium activity. Additionally, other compounds have been found
that have minimal activities in the mobilization of calcium from
bone while having significant activities in stimulating intestinal
calcium transport. Modification of the vitamin D side chain by
lengthening it at the 24-carbon has resulted in loss of calcium
activity and either an enhancement or undisturbed differentiative
activity. Placing the 24-methyl of 1.alpha.,25-dihydroxyvitamin
D.sub.2 in the epi-configuration appears to diminish activity in
the mobilization of calcium from bone. On the other hand, increased
hydrophobicity on the 26- and 27-carbons seems to increase the
total activity of the vitamin D compounds provided the 25-hydroxyl
is present.
Several of these known compounds exhibit highly potent activity in
vivo or in vitro, and possess advantageous activity profiles. Thus,
some of these compounds are in use, or have been proposed for use,
in the treatment of a variety of diseases such as renal
osteodystrophy, vitamin D-resistant rickets, osteoporosis,
psoriasis, and certain malignancies.
It is well known that females at the time of menopause suffer a
marked loss of bone mass giving rise ultimately to osteopenia,
which in turn gives rise to spontaneous crush fractures of the
vertebrae and fractures of the long bones. This disease is
generally known as postmenopausal osteoporosis and presents a major
medical problem, both in the United States and most other countries
where the life-span of females reaches ages of at least 60 and 70
years. Generally, the disease which is often accompanied by bone
pain and decreased physical activity, is diagnosed by one or two
vertebral crush fractures with evidence of diminished bone mass. It
is known that this disease is accompanied by diminished ability to
absorb calcium, decreased levels of sex hormones, especially
estrogen and androgen, and a negative calcium balance.
Similar symptoms of bone loss characterize senile osteoporosis and
steroid-induced osteoporosis, the latter being a recognized result
of long term glucocorticoid (cortico-steroid) therapy for certain
disease states.
Methods for treating the disease have varied considerably but to
date no totally satisfactory treatment is yet known. A conventional
treatment is to administer a calcium supplement to the patient.
However, calcium supplementation by itself has not been successful
in preventing or curing the disease. Another conventional treatment
is the injection of sex hormones, especially estrogen, which has
been reported to be effective in preventing the rapid loss of bone
mass experienced in postmenopausal women. This technique, however,
has been complicated by the fact of its possible carcinogenicity.
Other treatments for which variable results have been reported,
have included a combination of vitamin D in large doses, calcium
and fluoride. The primary problem with this approach is that
fluoride induces structurally unsound bone, called woven bone, and
in addition, produces a number of side effects such as increased
incidence of fractures and gastrointestinal reaction to the large
amounts of fluoride administered. Another suggested method is to
block bone resorption by injecting calcitonin or providing
phosphonates.
U.S. Pat. No. 4,225,596 suggests the use of various metabolites of
vitamin D.sub.3 for increasing calcium absorption and retention
within the body of mammals displaying evidence of or having a
physiological tendency toward loss of bone mass. The metabolites
specifically named in that patent, i.e., 1.alpha.-hydroxyvitamin
D.sub.3, 1.alpha.-hydroxyvitamin D.sub.2
1.alpha.,25-dihydroxyvitamin D.sub.3, 1.alpha.,25-dihydroxyvitamin
D.sub.2 and 1.alpha.,24,25-trihydroxyvitamin D.sub.3, although
capable of the activity described and claimed in that patent are
also characterized by the disadvantage of causing hypercalcemia
especially if used with the conventional calcium supplement
treatment. Therefore, use of these compounds to treat osteoporosis
has not been widely accepted. U.S. Pat. Nos. 3,833,622 and
3,901,928 respectively suggest using the hydrate of
25-hydroxyvitamin D.sub.3 and 1.alpha.-hydroxyvitamin D.sub.3 for
treatment of osteoporosis in a general expression of utility for
those compounds. It is well known both of those compounds express
traditional vitamin D-like activity, including the danger of
hypercalcemia.
U.S. Pat. No. 4,588,716 also suggests the use of
1.alpha.,25-dihydroxy-24-epi-vitamin D.sub.2 to treat bone
disorders characterized by the loss of bone mass, such as
osteoporosis. This compound expresses some of the vitamin D-like
characteristics affecting calcium metabolism such as increasing
intestinal calcium transport and stimulating the mineralization of
new bone. It also has the advantage of minimal effectiveness in
mobilizing calcium from bone. The 24-epi compound may be
administered alone or in combination with a bone mobilization
inducing compound such as a hormone or vitamin D compound such as
1.alpha.-hydroxyvitamin D.sub.3 or D.sub.2 or
1.alpha.,25-hydroxyvitamin D.sub.3 or D.sub.2.
U.S. Pat. No. 5,194,431 discloses the use of 24-cyclopropane
vitamin D.sub.2 compounds in treating osteoporosis. Also, U.S. Pat.
No. 4,851,401 discloses the use of cyclopentano
1,25-dihydroxyvitamin D.sub.3 compounds in the treatment of
osteoporosis and related diseases.
In an ongoing effort to develop a treatment for osteoporosis, and
to investigate the biological activity of vitamin D compounds, the
carbon 20 position of the side-chain was investigated to determine
its potential. Altering the order of substituents or the
substitution pattern on carbon 20 could result in a change of
minimum energy position for conformations around the C.sub.17
-C.sub.20 bond, and consequently, in a change of side-chain
orientation with respect to the ring system. Orientation of the
side-chain with respect to the ring system and configuration on the
C.sub.20 may have important consequences for biological properties
of cholestane derivatives, in particular vitamin D compounds. It is
well documented that binding of 1.alpha.,25-dihydroxyvitamin
D.sub.3 involves active centers in the ring A and triene system as
well as in the side-chain. Altering the "normal configuration"
around C.sub.17 -C.sub.20 bond in vitamin D could change the
distance between active centers within the molecule, and thus
result in a change in activity of such compounds.
SUMMARY OF THE INVENTION
The present invention provides novel (E)-20(22)-dehydrovitamin D
compounds exhibiting a desired, and highly advantageous, pattern of
biological activity. These compounds are characterized by a marked
intestinal calcium transport activity, as compared to that of
1.alpha.,25-dihydroxyvitamin D.sub.3, while exhibiting much lower
activity than 1.alpha.,25-dihydroxyvitamin D.sub.3 in their ability
to mobilize calcium from bone. Hence, these compounds are highly
specific in their calcemic activity. Their preferential activity on
intestinal calcium transport and reduced calcium mobilizing
activity in bone allows the in vivo administration of these
compounds for the treatment of metabolic bone diseases where bone
loss is a major concern. Because of their preferential calcemic
activity, these compounds would be preferred therapeutic agents for
the treatment of diseases where bone formation is desired, such as
osteoporosis, osteomalacia and renal osteodystrophy. These
compounds also have relatively high HL-60 cell differentiation
activity which makes them particularly suitable for use in treating
diseases characterized by abnormal cell differentiation and/or cell
proliferation, such as psoriasis.
Structurally, the key feature of the compounds having these
desirable biological attributes is that they are analogs of vitamin
D.sub.3 in which a double bond has been introduced into the
side-chain between carbons 20 and 22. Thus, the compounds of this
type are characterized by structures I and II: ##STR1## where
Y.sup.1 may be hydrogen or a hydroxy-protecting group, R.sup.1
represents hydrogen, hydroxy or protected hydroxy, and where R is
hydrogen, aryl, alkyl, hydroxyalkyl or fluoroalkyl group, or R may
represent the following side chain fragment: ##STR2## wherein
R.sup.1 is as defined above, R.sup.2 and R.sup.3 are each selected
from the group consisting of alkyl, hydroxyalkyl and fluoroalkyl,
or, when taken together represent the group --(CH.sub.2).sub.m --
where m is an integer having a value of from 2 to 5, R.sup.4 is
selected from the group consisting of hydrogen, hydroxy, fluorine,
O-acyl, alkyl, hydroxyalkyl and fluoroalkyl, R.sup.5 is selected
from the group consisting of hydrogen, fluorine, alkyl,
hydroxyalkyl and fluoroalkyl, and wherein n is an integer having a
value of from 1 to 5.
The present invention, therefore, provides novel compounds showing
preferential activity on intestinal calcium transport and reduced
calcium mobilizing activity in bone, and are useful for the
treatment of metabolic bone diseases, such as osteoporosis, where
bone loss is a major concern. High cell differentiation activity
also makes them suitable for treating diseases characterized by
abnormal cell differentiation and/or cell proliferation, such as
psoriasis. More specifically, the preferred compound is
(E)-20(22)-dehydro-1.alpha.,25 dihydroxyvitamin D.sub.3.
This invention also provides novel intermediate compounds formed
during the synthesis of the end products. Structurally, the
intermediate compounds are characterized by the following general
structure: ##STR3## where Y.sup.1 and R.sup.1 are as previously
defined herein.
Other key intermediates are characterized by the following general
structures: ##STR4## where Y.sup.1 and R.sup.1 are as previously
defined herein.
In another aspect of the invention, it has now been found that the
loss of bone mass, which is characteristic of osteoporosis may be
effectively treated by the administration of a
(E)-20(22)-dehydrovitamin D compound in sufficient mounts to
increase bone mass. More specifically, a method of treating
osteoporosis comprises the administration of an effective amount of
a (E)-20(22)-dehydrovitamin D compound, preferably
(E)-20(22)-dehydro-1.alpha.,25-dihydroxyvitamin D.sub.3. The above
compounds may be administered alone or in combination with other
pharmaceutically acceptable agents. Dosages of from not less than
about 0.5 .mu.g/day to not more than about 50 .mu.g/day of the
individual compound per se, or in combinations, are generally
effective. This method has the distinct advantage that it will
restore bone mass due to the preferential intestinal activity and
the insignificant bone mobilization activity of these compounds.
Further, these compounds advantageously will not cause
hypercalcemia even if the compound is administered continuously on
a daily basis, as long as the appropriate compound dosages are
used, it being understood that the dosage levels will be adjusted
dependent on the response of the subject as monitored by methods
known to those skilled in the art.
The above method, involving the administration of the indicated
dosages of (E)-20(22)-dehydrovitamin D compounds such as
(E)-20(22)-dehydro-1.alpha.,25-dihydroxyvitamin D.sub.3 is
effective in restoring or maintaining bone mass, and thus provides
a novel method for the treatment or prevention of various forms of
osteoporosis such as postmenopausal osteoporosis, senile
osteoporosis and steroid-induced osteoporosis. It will be evident
that the method will find ready application for the prevention or
treatment of disease states other than those named, in which the
loss of bone mass is an indication.
In still another aspect of the invention, it has now been found
that abnormal cell differentiation and/or cell proliferation, which
is characteristic of diseases such as psoriasis, may be effectively
treated by the administration of a (E)-20(22)-dehydrovitamin D
compound in sufficient amounts to treat the disease. More
specifically, a method of treating psoriasis comprises the
administration of an effective amount of a
(E)-20(22)-dehydrovitamin D compound, preferably
(E)-20(22)-dehydro-1.alpha.,25-dihydroxyvitamin D.sub.3. The above
compounds may be administered alone or in combination with other
pharmaceutically acceptable agents. Dosages of from not less than
about 0.5 .mu.g/day to not more than about 50 .mu.g/day of the
individual compound per se, or in combinations, are generally
effective. This method has the distinct advantage that it will
treat the disease and further these compounds advantageously will
not cause hypercalcemia even if the compound is administered
continuously on a daily basis, as long as the appropriate compound
dosages are used, it being understood that the dosage levels will
be adjusted dependent on the response of the subject as monitored
by methods known to those skilled in the art.
DETAILED DESCRIPTION OF THE INVENTION
In this specification and the claims, the term "hydroxy-protecting
group" refers to any group commonly used for the protection of
hydroxy functions during subsequent reactions, including, for
example, acyl or alklsilyl groups such as trimethylsilyl,
triethylsilyl, t-butyldimethylsilyl and analogous alkylated silyl
radicals, or alkoxyalkyl groups such as methoxymethyl,
ethoxymethyl, methoxyethoxymethyl, tetrahydrofuranyl or
tetrahydropyranyl. A "protected-hydroxy" is a hydroxy function
derivatized by one of the above-hydroxy-protecting groupings.
"Alkyl" signifies a straight-chain or branched hydrocarbon radical
of 1 to 10 carbons in all its isomeric forms, such as methyl,
ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, etc., and the
terms "hydroxyalkyl" and "flouoroalkyl" refer to such an alkyl
radical substituted by one or more hydroxy or fluoro groups
respectively. The term "acyl" means an alkanoyl group of 1 to 6
carbons in all its isomeric forms, such as formyl, acetyl,
propionyl, etc. or an aroyl group, such as benzoyl, nitrobenzoyl or
halobenzoyl, or a dicarboxylic acyl group such as oxalyl, malonyl,
succinoyl, glutaroyl, or adipoyl. The term "aryl" signifies a
phenyl-, or an alkyl-, nitro- or halo-substituted phenyl group. A
wavy line to the substituents at C-20 and C-22 indicate that these
substituents may have either the R or S configuration.
The vitamin D compounds useful in the present treatment are
(E)-20(22)-dehydrovitamin D compounds, preferably
(E)-20(22)-dehydro-1.alpha.,25-dihydroxyvitamin D.sub.3. The above
compounds may be administered alone or in combination with other
pharmaceutically acceptable agents.
The (E)-20(22)-dehydrovitamin D compounds or combinations thereof
can be readily administered as sterile parenteral solutions by
injection or intravenously, or by alimentary canal in the form of
oral dosages, or transdermally, or by suppository or by topical
formulations. Doses of from about 0.5 micrograms to about 50
micrograms per day of the compounds per se, or in combination with
other 1.alpha.-hydroxylated vitamin D compounds, the proportions of
each of the compounds in the combination being dependent upon the
particular disease state being addressed and the degree of bone
mineralization and/or bone mobilization desired, are generally
effective to practice the present invention. In all cases
sufficient amounts of the compound should be used to restore bone
mass. Amounts in excess of about 50 micrograms per day or the
combination of that compound with other 1.alpha.-hydroxylated
vitamin D compounds, are generally unnecessary to achieve the
desired results, may result in hypercalcemia, and may not be an
economically sound practice. In practice the higher doses are used
where therapeutic treatment of a disease state is the desired end
while the lower doses are generally used for prophylactic purposes,
it being understood that the specific dosage administered in tony
given case will be adjusted in accordance with the specific
compounds being administered, the disease to be treated, the
condition of the subject and the other relevant medical facts that
may modify the activity of the drug or the response of fine
subject, as is well known by those skilled in the art. For example,
to be effective, the
(E)-20(22)-dehydro-1.alpha.,25-dihydroxyvitamin D.sub.3 compound is
preferably administered in a closage range of 0.5-50 .mu.g/day. In
general, either a single daily dose or divided daily dosages may be
employed, as is well known in the art.
Dosage forms of the various compounds can be prepared by combining
them with non-toxic pharmaceutically acceptable carriers to make
either immediate release or slow release formulations, as is well
known in the art. Such carriers may be either solid or liquid such
as, for example, corn starch, lactose, sucrose, peanut oil, olive
oil, sesame oil and propylene glycol. If a solid carrier is used
the dosage form of the compounds may be tablets, capsules, powders,
troches or lozenges. If a liquid carrier is used, soft gelatin
capsules, or syrup or liquid suspensions, emulsions or solutions
may be the dosage form. The dosage forms may also contain
adjuvants, such as preserving, stabilizing, wetting or emulsifying
agents, solution promoters, etc. They may also contain other
therapeutically valuable substances.
Formulations suitable for topical administration include liquid or
semi-liquid preparations such as liniments, lotions, applicants,
oil-in-water or water-in-oil emulsions such as creams, ointments or
pastes; or solutions or suspensions such as drops; or as
sprays.
The topical compositions of this invention are formulated
preferably as creams, lotions, ointments and the like by choice of
appropriate carriers. Suitable carriers include vegetable or
mineral oils, white petrolatum (white soft parafin), branched chain
fats or oils, animal fats and high molecular weight alcohol
(greater than C.sub.12). The preferred carriers are those in which
the active ingredient is soluble. Emulsifiers, stabilizers and
antioxidants may also be included as well as agents imparting color
or fragrance if desired.
Topical creams are preferably formulated from a mixture of mineral
oil, self-emulsifying beeswax and water in which mixture the active
ingredient, dissolved in a small amount of an oil such as almond
oil, is admixed. A typical example of such a cream is one which
includes about 39 parts water, about 20 parts beeswax, about 40
parts mineral oil and about 1 part almond oil.
Topical ointments may be formulated by mixing a solution of the
active ingredient in a vegetable oil such as almond oil with warm
soft paraffin and allowing the mixture to cool. A typical example
of such an ointment is one which includes about 30% almond oil and
about 70% white soft paraffin by weight.
Topical lotions may be conveniently prepared by dissolving the
active ingredient, in a suitable high molecular weight alcohol such
as propylene glycol or polyethylene glycol.
The present invention is more specifically described by the
following examples, which are meant to be illustrative only of the
process of synthesis and of the novel compounds, both end products
and intermediates, obtainable thereby. In these examples, specific
con:pounds identified by Arabic numerals (e.g. compounds 1, 2, 3, .
. . etc.) refer to the structures so numbered in the process
schematics. Additionally examples are provided which are
illustrative of the distinctive biological characteristics of the
new compounds, such characteristics serving as a basis for the
application of these compounds in the treatment of metabolic bone
disease and psoriasis.
PREPARATION OF COMPOUNDS
The preparation of 20(22)-dehydrovitamin analogs having the basic
structures shown above can be accomplished starting from the
diene-protected derivatives of structures III and IV or ##STR5##
vitamin D compounds of the general structures V through VIII where
Y.sup.1 and R.sup.1 awe as defined above. ##STR6## Reaction of the
C-22 aldehydes III, V and VI with the alkylmagnesium halide having
the structure RMgX (R as defined above, X=halogen) or alkyllithium
reagent having the structure RLi (R as defined above) in the
appropriate inert solvent, provides 22-hydroxy compounds of the
general formulas IX, X and XI (Y.sup.2 =H).
The 22-hydroxy intermediates IX, X and XI (Y.sup.2 =H) having all
remaining hydroxy groups protected, can be then directly dehydrated
to the 20(22)-dehydro analogs XII, I and II, respectively. ##STR7##
It is well known that dehydration processes of the C-20 alcohols
usually give a mixture of isomeric side-chain olefins [(see for
example W. R. Ness et al., J. Org. Chem., 41, 3429 (1976)]. It can
be therefore advantageous to subject the hydroxy compounds IX, X
and XI to the reaction with an alkyl- or arylsulfonylhalide (e.g.
methanesulfonyl chloride, p-toluenesulfonyl chloride) in a suitable
solvent (e.g. pyridine) and obtain the corresponding 22-O-alkyl- or
arylsulfonyl derivatives (the compounds having the structures shown
IX, X or XI above, where y.sup.2 is alkyl-SO.sub.2 -- or
aryl-SO.sub.2 --). These compounds are then subjected to the
appropriate reaction conditions which promote sulfonate ester 1,2
elimination process, such as treatment with NaJ, reaction with
pyridine, DBU, DBN or another base, reaction on alumina (Al.sub.2
O.sub.3) column etc. These elimination reactions provide
satisfactory yield of (E)-20(22)-compounds I, II and XII. Adduct
XII subjected to basic conditions can be then converted to
5,7-diene steroid which in turn, via the well known process
consisting of irradiation with UV light and thermal isomerization,
gives I.
Alternatively, the 20-ketones IV, VII and VIII can be subjected to
Wittig (or Wittig-Horner) reaction with the alkylide-nephophoranes
generated from triphenylphosphonium salt having the general formula
##STR8## or the phosphinoxy carbanion prepared from the phosphine
oxide ##STR9## (R as defined above, X=halogen) to obtain the
corresponding (E)-20(22)-dehydro compounds XII, I and II,
respectively. It is well known that Wittig-type reactions with
20-keto steroids afford exclusively 20(22)-unsaturated products
with the (E) configuration [see for example S. R. Show et al, J.
Org. Chem., 44, 3760 (1979)].
The next step of the process comprises the removal of the
hydroxy-protecting groups to produce the free hydroxy compounds
represented by vitamin D structures I and II above (where Y.sup.1
is hydrogen and R.sup.1 can be hydrogen or hydroxyl). If desired,
the 5,6-cis compounds I, V, VII and X can be easily converted to
the corresponding 5,6-trans counterparts (and vice versa) II, IV,
VIII and XI, respectively, by the known iodide-catalyzed
isomerization process [see A. Verloop et al., Rec. Trav. Chim.
Pays-Bas 78, 1004 (1959)].
EXAMPLE I
Reaction of PTAD-protected diene-aldehyde 1 with Grignard reagent
derived from bromocompound A (Scheme I)
A solution of the known 4-bromo-2-methyl-2-triethylsily-loxy)butane
A (281 mg, 1 mmol) in anhydrous ether (0.5 mL, containing a
catalytic quantity of iodine) was added dropwise to a stirred
mixture of magnesium powder (29 mg, 1.2 mmol; .about.50 mesh,
Aldrich) in anhydrous ether (0.5 mL) under argon at room
temperature with occasional warming it up to 35.degree. C. After
addition was complete the mixture was stirred for 1 h at room
temperature and for 30 min at 40.degree. C. Then it was cooled at
0.degree. C. and a solution of a known PTAD-protected diene
(20S)-C-22 aldehyde 1 (123 mg, 0.2 mmol) in anhydrous THF (0.5 mL,
cooled to 0.degree.C.) was added dropwise. After the mixture was
stirred for 20 min at 0.degree. C. and 1 h at room temperature it
was quenched with aqueous solution of NH.sub.4 Cl (2 mL) and
diluted with benzene (20 mL). The organic layer was separated,
washed with brine and diluted NaHCO.sub.3, dried (Na.sub.2
SI.sub.4) and evaporated. Flash chromatography of the residue using
20% ethyl acetate in hexane as an eluent yielded pure (22S)-alcohol
2 (135 mg, 83%) as a foam: .sup.1 H-NMR (CDCl.sub.3, 500 MHz):
.delta.0.084 and 0.106 (3H and 3H, each s, 2.times.SiMe), 0.578
(6H, q, J=8 Hz, 3.times.SiCH.sub.2), 0.813 (3H, s, 18-H.sub.3),
0.885 (9H, s, Si-t-Bu), 0.936 (3H, d, J=7.7 Hz, 21-H.sub.3), 0.944
(9H, t, J=8 Hz, 3.times.SiCH.sub.2 CH.sub.3), 0.968 (3H, s,
19-H.sub.3), 1.222 (6H, br s, 26- and 27-H.sub.3), 3.12 (1H, dd,
J.sub.1 =14.2 Hz, J.sub.2 =5.1 Hz, 9.alpha.-H), 3.65 (1H, m, 22-H),
4.40 (1H, br m, 3.alpha.-H), 6.20 and 6.38 (2H, each d, J=8.2 Hz,
6- and 7-H), 7.3-7.5 (5H, br m, Ar--H); MS m/z (rel intensity) 819
(M.sup.+, 19), 762 (48), 644 (M.sup.+ -RDA, 74), 497 (61), 119
(PhNCO, 100).
EXAMPLE 2
Reaction of 22-hydroxy PTAD-protected-diene 2 with
p-toluenesulfonyl chloride
To a solution of alcohol 2 (75 mg, 0.093 mmol) in dry pyridine (200
.mu.l) was added fleshly recrystallized p-toluenesulfonyl chloride
(49 mg, 0.26 mmol) and the reaction was allowed to proceed for 64 h
at 4.degree. C. The reaction mixture was poured into ice/saturated
NaHCO.sub.3 with stirring. After 40 min. of stirring the aqueous
suspension was extracted with 1:1 (v/v) benzene/ether (3.times.10
mL). The combined extracts were washed with saturated NaHCO.sub.3,
water, saturated CuSO.sub.4, again water, dried (Na.sub.2 SO.sub.4)
and evaporated. The oily yellowish residue (quantitative yield) was
pure enough to be used for the subsequent synthetic step.
Analytical sample of the tosylate 3 was obtained after HPLC
purification (Zorbax-Silica column 6.2 mm.times.25 cm) using 10%
ethyl acetate in hexane (R.sub.v 16 mL): .sup.1 H-NMR (CDCl.sub.3,
500 MHz): .delta.0.089 and 0.108 (3H and 3H, each s, 2.times.
SiMe), 0.537 (6H, q, J=7.9 Hz, 3.times.SiCH.sub.2), 0.723 (3H, s,
18-H.sub.3), 0.887 (9H, s, Si-t-Bu), 0.921 (9H, t, J=7.9 Hz,
3.times.SiCH.sub.2 CH.sub.3), .about.0.94 (3H, d, J=7 Hz,
21-H.sub.3), 0.943 (3H, s, 19-H.sub.3), 1.123 auld 1.157 (3H and
3H, each s, 26- and 27-H.sub.3), 2.43 (3H, s, Ar-Me), 3.13 (1H, dd,
J.sub.1 =14.2 Hz, J.sub.2 =5.0 Hz, 9.alpha.-H), 4.39 (1H, br m,
3.alpha.-H), 4.49 (1H, t, J=7.0 Hz, 22-H), 6.18 and 6.30 (2H, each
d, J=8.3 Hz, 6- and 7-H), 7.3-7.5 (7H, br m, Ar--H), 7.79 (2H, d,
J=8.2 Hz, Ar--H).
EXAMPLE 3
Reaction of 22-tosyloxy PTAD-protected diene 3 with sodium
iodide
To a stirred solution of tosylate 3 (4.9 mg, 5 .mu.mol) in 1:1
(v/v) acetone/2-butanone (200 .mu.L) was added calcium carbonate (1
mg, 10 .mu.mol) followed by sodium iodide (3.7 mg, 25 .mu.mol). The
resultant mixture was stirred and heated for 80 h at 55.degree. C.
under argon, by which time no starting material remained. The
reaction mixture was poured into water (10 mL) and extracted with
ethyl acetate (2.times.10 mL). The combined organic layers were
washed with 1% Na.sub.2 SO.sub.3 and water, dried (Na.sub.2
SO.sub.4) and evaporated. The residue was separated by preparative
HPLC (6.2 mm.times.25 cm Zorbax-Silica column) using 3% ethyl
acetate in hexane as an eluent. Pure (E)-20(22)-dehydro compound 4
(2.2 mg, 55%; collected at 52 mL) was obtained as a foam: .sup.1
H-NMR (CDCl.sub.3, 500 MHz): .delta.0.087 and 0.109 (3H and 3H,
each s, 2.times.SiMe), 0.571 (6H, q, J=8 Hz, 3.times.SiCH.sub.2),
0.658 (3H, s, 18-H.sub.3), 0.888 (9H, s, Si-t-Bu), 0.950 (9H, s,
26- and 27-H.sub.3), 1.639 (3H, s, 21-H.sub.3), 3.13 (1H, dd,
J.sub.1 =14.1 Hz, J.sub.2 =5.1 Hz, 9.alpha.-H), 4.40 (1H, br m,
3.alpha.-H), 5.23 (1H, t, J=7.0 Hz, 22-H), 6.19 and 6.36 (2H, each
d, J=8.3 Hz, 6- and 7-H), 7.3-7.5 (5H, br m, Ar--H); MS m/z (rel
intensity) 801 (M.sup.+, <1), 626 (M.sup.+ -RDA, 100), 479 (77),
119 (PhNCO, 58).
Compound 4 is a direct precursor of the (E)-20(22)-dehydrovitamin
D.sub.3.
EXAMPLE 4
Elimination of p-toluenesulfonate ester 3 in pyridine
A solution of tosylate 3 (1 mg, 1 .mu.mol) in a dry pyridine (200
.mu.l) was heated under argon for 48 h at 70.degree. C. Solvent was
evaporated, the residue taken up in ethyl acetate and the solution
was washed with saturated CuSO.sub.4, water and saturated
NaHCO.sub.3, dried (Na.sub.2 SO.sub.4) and evaporated. HPLC
separation of the residue (6.2 mm.times.25 cm Zorbax-Silica column)
using 3% ethyl acetate in hexane yielded the olefin 4 (0.29 mg,
35%; collected at 52 mL).
EXAMPLE 5
Reaction of vitamin D C-22 aldehyde 5 with Grignard reagent derived
from bromocompound A
A solution of 4-bromo-2-methyl-2-triethylsilyloxy)butane A (94 mg,
0.33 mmol) in anhydrous ether (0.3 mL) was added dropwise to a
stirred mixture of magnesium powder (9.7 mg, 0.4 mol; .about.50
mesh, Aldrich) in anhydrous ether (0.2 mL) under argon at room
temperature with occasional warming it up to 35.degree. C. After
addition was complete the mixture was stirred for 15 min. at room
temperature and 30 min. at 40.degree. C. Then it was cooled to
0.degree. C. and a solution of a known (20S)-C-22 vitamin aldehyde
5 [(32 mg, 0.056 mmol; see A. Kutner et al., J. Org. Chem. 53, 3450
(1988)] in annydrous ether (0.3 mL, cooled to 0.degree. C.) was
added dropwise. After the mixture was stirred for 20 min at
0.degree. C. and 75 min. at room temperature it was quenched with
aqueous solution of NH.sub.4 Cl (2 mL) and diluted with 4:1 (v/v)
benzene/ether (20 mL). The organic layer was separated, washed with
brine and diluted NaHCO.sub.3, dried (Na.sub.2 SO.sub.4) and
evaporated. TLC and HPLC control indicated formation of only one
(22S) of the two possible isomers. Pure (22S)-hydroxyvitamin D
derivative 6 was obtained as a colorless oil (31 mg, 75%) by
preparative HPLC (Zorbax-Silica column 6.2 mm.times.25 cm) using
3.5% ethyl acetate in hexane as an eluent; peak at 30 mL was
collected. UV (EtOH) .lambda..sub.max 264 nm, .lambda..sub.min 225
nm, A.sub.265 /A.sub.265 =1.6; .sup.1 H-NMR (CDCl.sub.3, 500 MHz):
.delta.0.062 (12H, br s, 4.times.SiMe), 0.546 (3H, s, 18-H.sub.3),
0.583 (6H, q, J=8 Hz, 3.times.SiCH.sub.2), 0.887 (18H, s,
2.times.Si-t-Bu), 0.920 (3H, d, J=6.4 Hz, 21-H.sub.3), 0.947 (9 H,
t, J=8 Hz, 3.times.SiCH.sub.2 CH.sub.3), 1.221 and 1.225 (3H and
3H, each s, 26- and 27-H.sub.3), 2.83 (1H, br d, J=12.5 Hz,
9.beta.-H), 3.63 (1H, m, 22-H), 4.19 (1H, m, 3.alpha.-H), 4.37 (1H,
m, 1.beta.-H), 4.87 and 5.18 (1H and 1H, each s, 19-H.sub.2), 6.02
(1H, d, J=11.2 Hz, 7-H), 6.24 (1H, d, J=11.2 Hz, 6-H); MS m/z (rel
intensity) 774 (M.sup.+, 15) 642 (43) 75 (100); exact mass called
for C.sub.45 H.sub.86 O.sub.4 Si.sub.3 774.5834, found
774.5850.
EXAMPLE 6
Reaction of 22-hydroxyvitamin D compound 6 with p-toluenesulfonyl
chloride
To a solution of alcohol 6 (28 mg, 0.036 mmol) in dry pyridine (100
.mu.l ) was added fleshly recrystallized p-toluenesulfonyl chloride
(20 mg, 0.10 mmol) and the reaction was allowed to proceed for 64 h
at 4.degree. C. The reaction mixture was poured into ice/saturated
NaHCO.sub.3 with stirring. After 40 min. of stirring the aqueous
suspension was extracted with 4:1 (v/v) benzene/ether (3.times.10
mL). The combined extracts were washed with saturated NaHCO.sub.3,
water, saturated CuSO.sub.4, again water, dried (Na.sub.2 SO.sub.4)
and evaporated. The oily yellowish residue was purified by
preparative HPLC (Zorbax-Silica column 6.2 mm.times.25 cm) using 2%
ethyl acetate in hexane as an eluent. Pure tosylate 7 was obtained
as a colorless oil: UV (hexane) .lambda..sub.max 264 and 223 nm,
.lambda..sub.min 238 nm; .sup.1 H-NMR (CDCl.sub.3, 500 MHz):
.delta.0.059 and 0.067 (6H and 6H, each s, 2.times.SiMe.sub.2),
0.479 (3H, s, 18-H.sub.3), 0.528 (6H, q, J=8 Hz,
3.times.SiCH.sub.2), 0.877 (18H, s, 2.times.Si-t-Bu), 0.915 (9H, t,
J=8 Hz, 3.times.SiCH.sub.2 CH.sub.3), 0.929 (3H, d, J=6.0 Hz,
21-H.sub.3), 1.103 and 1.141 (3H and 3H, each s, 26- and
27-H.sub.3), 2.43 (3H, s, Ar-Me), 2.80 (1H, br d, J=12.3 Hz,
9.beta.-H), 4.19 (1H, m, 3.alpha.-H), 4.38 (1H, m, 1.beta.-H), 4.58
(1H, t, J=7.1 Hz, 22-H), 4.86 and 5.19 (1H and 1H, each s,
19-H.sub.2), 5.99 (1H, d, J=11.2 Hz, 7-H), 6.22 (1H, d, J=11.2 Hz,
6-H), 7.32 (2H, d, J=8 Hz, Ar--H), 7.80 (2H, d, J= 8 Hz, Ar--H); MS
m/z (rel intensity) 928 (M.sup.+, 1), 796 (2), 756 (3), 664 (3),
624 (47), 492 (27), 173 (100); exact mass calcd for C.sub.52
H.sub.92 O.sub.6 Si.sub.3 S 928.5922, found 928.5894.
EXAMPLE 7
Reaction of vitamin D 22-p-toluenesulfonate 7 with sodium
iodide
To a stirred solution of tosylate 7 (4.6 mg, 5 .mu.mol) in 1:1
(v/v) acetone/2-butanone (200 .mu.L) was added calcium carbonate (1
mg, 10 .mu.mol) followed by sodium iodide (3.7 mg, 25 .mu.mol). The
resultant mixture was stirred and heated for 100 h at 45.degree. C.
in the dark under argon, by which time no stating material
remained. The reaction mixture was poured into water (10 mL) and
extracted with ethyl acetate (2.times.10 mL). The combined organic
layers were washed with 1% Na.sub.2 SO.sub.3 and water, dried
(Na.sub.2 SO.sub.4) and evaporated. The work up of the reaction
mixture and following chromatographic separations were done using
subdued light in the laboratory. The mixture of products was
separated by preparative HPLC (6.2 mm.times.25 cm Zorbax-Silica
column) using 0.1% ethyl acetate in hexane as an eluent. The main
product 8 (peak at R.sub.v 34 mL) was isolated and repurified by
HPLC in the same solvent system (recycling mode) to give an
analytically pure material (1.2 mg, 32%): UV (hexane)
.lambda..sub.max 265.0 nm, .lambda..sub.min 228.5 nm (A.sub.265
/A.sub.228 =1.7); .sup.1 H-NMR (CDCl.sub.3, 500 MHz): .delta.0.063
and 0.072 (6H and 6H, each s, 2.times.SiMe.sub.2), 0.403 (3H, s,
18-H.sub.3), 0.567 (6H, q, J=8 Hz, 3.times.SiCH.sub.2), 0.884 (18H,
s, 2.times.Si-t-Bu), 0.946 (9H, t, J=8 Hz, 3.times.SiCH.sub.2
CH.sub.3), 1.264 (6H, br s, 26- and 27-H.sub.3), 1.630 (3H, s,
21-H.sub.3), 2.84 (1H, br d, J=12.8 Hz, 9.beta.-H), 4.19 (1H, m,
3.alpha.-H), 4.38 (1H, m, 1.beta.-H), 4.87 (1H, s, one of
19-H.sub.2), 5.19 (2H, m, one of 19-H.sub.2 and 22-H), 6.02 (1H, d,
J=11.2 Hz, 7-H), 6.24 (1H, d, J=11.2 Hz, 6-H); MS m/z (rel
intensity) 756 (M.sup.+, 12), 624 (31), 248 (47), 117 (100); exact
mass calcd for C.sub.45 H.sub.84 O.sub.3 Si.sub.3 756.5728, found
756.5707.
EXAMPLE 8
Deprotection of hydroxyl groups in (E)-20(22)-dehydro compound
8
To a solution of protected triol 8 (0.93 mg, 1.22 .mu.mol) in
anhydrous benzene (40 .mu.L) was added AG 50W-X.sub.4 ion exchange
resin (15 mg, prewashed with methanol) as a slurry in anydrous
methanol (200 .mu.L). The mixture was vigorously stirred at room
temperature for 13 h under argon, and it was diluted with 1:1 (v/v)
ether/ethyl acetate (1 mL). The solution was decanted and
transferred to separatory funnel and the resin was washed with 1:1
ether/ethyl acetate (2.times.2 mL). The combined organic phase was
washed with 5 nL portions of brine, 1% Na.sub.2 S.sub.2 O.sub.3,
saturated NaHCO.sub.3, and brine again, dried (Na.sub.2 SO.sub.4)
and evaporated. Purification of product by HPLC (6.2 mm.times.25 cm
Zorbax-Silica column) using 1:1 (v/v) hexane/ethyl acetate as an
eluent provided crystalline triol 9 (337 .mu.g, 66%; eluted at
R.sub.v 59 mL): UV (EtOH) .lambda. .sub.max 265.0 nm,
.lambda..sub.min 228.5 nm (A.sub.265 /A.sub.228 =1.8); .sup.1 H=NMR
(CDCl.sub.3, 500 MHz): .delta.0.424 (3H, s, 18-H.sub.3), 1.231 (6H,
br s, 26- and 27-H.sub.3), 1.651 (3H, s, 21-H.sub.3), 2.84 (1H, br
d, J=12.2 Hz, 9.beta.-H), 4.23 (1H, m, 3.alpha.-H), 4.44 (1H, m,
1.beta.-H), 5.00 (1H, s, one of 19-H.sub.2), 5.23 (1H, t, J=7.1 Hz,
22-H), 5.33 (1H, s, one of 19-H.sub.2), 6.03 (1H, d, J=11.3 Hz,
7-H), 6.38 (1H, d, J=11.3 Hz, 6-H); MS m/z (rel intensity) 414
(M.sup.+, 8), 396 (M.sup.+ -) H.sub.2 O, 15), 378 (M.sup.+ -2
H.sub.2 O, 10), 152 (37), 134 (100); exact mass calcd for C.sub.27
H.sub.42 O.sub.3 414.3134, found 414.3138. ##STR10## Biological
Activity
Rats were maintained on a normal calcium and normal phosphorus diet
for one week (0.47% Ca, 0.3% P), then switched to a --Ca diet for
the duration of the experiment (0.02% Ca). Vitamin D compounds were
suspended in mixtures of ethanol and propylene glycol (5%:95%) and
were administered daily for 6 days intraperitoneally.
After 6 days the rats were killed and the duodena were used for
determination of intestinal calcium transport by the everted
intestinal sac technique (Martin & DeLuca, 1967) and serum
calcium (bone calcium mobilization). The tests were made against
the 1,25-dihydroxyvitamin D.sub.3 standard and are reported in
Table 1.
TABLE 1 ______________________________________ INTESTINAL CALCIUM
TRANSPORT AND BONE CALCIUM MOBILIZING ACTIVITIES OF
(E)-20(22)-DEHYDRO-1.alpha.,25-DIHYDROXYVITAMIN D.sub.3 Amount
Serum Ca (.mu.gs/d6 S/M (ave.s.e.m.) Compound days) (ave.s.e.m.)
(mg %) ______________________________________ D-Deficient 0 3.98
+/- 0.19 4.14 +/- 0.07 1.alpha.,25-(OH).sub.2 D.sub.3 0.1 .mu.g/d
11.2 +/- 1.2 5.9 +/- 0.27 (E)-20(22)-dehydro- 1 .mu.g/d 12.4 +/-
0.73 5.2 +/- 0.23 1.alpha.,25-(OH).sub.2 D.sub.3
______________________________________
The results show that the (E)-20(22)-dehydro-1,25-dihydroxyvitamin
D.sub.3 compound is less active than 1,25-dihydroxyvitamin D.sub.3
in mobilization of calcium from bone since the serum calcium levels
of the compound are less than that found for
1.alpha.,25-dihydroxyvitamin D.sub.3 even though the dosage is 10
times greater. However, the
(E)-20(22)-dehydro-1,25-dihydroxyvitamin D.sub.3 compound has
highly significant intestinal calcium transport activity. These
compounds therefore, by showing preferential activity on intestinal
calcium transport and reduced calcium mobilizing activity in bone
suggest that they are preferred agents for the treatment of a
disease where bone loss is a major issue, such as osteoporosis,
osteomalacia and renal osteodystrophy.
Measurement Of Differentiation in HL-60 Cells
The measurement of differentiation in HL-60 cells (human leukemia
cells) was carried out according to the general procedures
described by DeLuca et al., U.S. Pat. No. 4,717,721. As shown in
Table 2, degree of differentiation is assessed by a standard assay,
namely, NBT reduction, and results are expressed as the percent of
differentiated cells produced in response to treatment with various
concentrations of vitamin D compounds.
TABLE 2 ______________________________________ Differentiation
Activity in HL-60 Cells in Culture % Cells Showing Differentiation
Compound Concentration (molar) NBT Reduction
______________________________________ 1.25-(OH).sub.2 D.sub.3 1
.times. 10.sup.-7 90 .+-. 2 5 .times. 10.sup.-8 69 .+-. 3 1 .times.
10.sup.-8 58 .+-. 3 1 .times. 10.sup.-9 36 .+-. 2
.DELTA..sup.20(22) -1,25(OH).sub.2 D.sub.3 1 .times. 10.sup.-7 85
.+-. 3 5 .times. 10.sup.-8 67 .+-. 3 1 .times. 10.sup.-8 34 .+-. 2
1 .times. 10.sup.-9 12 .+-. 2
______________________________________
The results of this assay is shown in Table 2. It is evident that
the novel analogs are about equally as active as 1,25-(OH).sub.2
D.sub.3 itself in causing differentiation of HL-60 cells in
culture. For treatment purposes, the novel compounds of this
invention may be formulated for pharmaceutical applications as a
solution in innocuous solvents, or as an emulsion, suspension or
dispersion in suitable solvents or carriers, or as pills, tablets
or capsules together with solid carriers, according to conventional
methods known in the art. Any such formulations may also contain
other pharmaceutically-acceptable and non-toxic excipients such as
stabilizers, anti-oxidants, binders, coloring agents or emulsifying
or taste-modifying agents.
The compounds may be administered orally, topically, parenterally
or transdermally. The compounds are advantageously administered by
injection or by intravenous infusion of suitable sterile solutions,
or in the form of liquid or solid doses via the alimentary canal,
or in the form of creams, ointments, patches, or similar vehicles
suitable for transdermal applications. Doses of from 0.5 .mu.g to
50 .mu.g per day of the compounds are appropriate for treatment
purposes, such doses being adjusted according to the disease to be
treated, its severity and the response of the subject, as is well
understood in the art. Since the new compounds exhibit specificity
of action, each may be suitably administered alone, in situations
where only calcium transport stimulation is desired, or together
with graded doses of another active vitamin D compound--e.g.
1.alpha.-hydroxyvitamin D.sub.2 or D.sub.3, or
1.alpha.,25-dihydroxyvitamin D.sub.3 --in situations where some
degree of bone mineral mobilization (together with calcium
transport stimulation) is found to be advantageous.
Various modes of carrying out the invention are contemplated as
being within the scope of the following claims particularly
pointing out and distinctly claiming the subject matter regarded as
the invention.
* * * * *